Aims: We recently reported the alveolar wall liquid (AWL), the liquid layer adjacent to the alveolar epithelium is established by Cl--dependent liquid secretion from alveolar epithelial cells (AEC). The alveolar cystic fibrosis transmembrane regulator (CFTR) regulates this secretion. Here our overall objective is to understand factors regulating AWL secretion. In the three specific aims, we will test the hypotheses that (1) alveolar type I (AT1) but not type 2 (AT2) establish AWL secretion (2) in hypoxia, hydrogen peroxide (H2O2) blocks AWL secretion, and that (3) in lung inflammation, nitric oxide (NO) blocks AWL secretion. Procedures: In all specific aims, studies will be developed through two-photon microscopy of isolated mouse lungs. We will determine AWL secretion through real-time fluorescent imaging and determine signaling pathways regulating AWL secretion by immunostaining, pharmacological inhibition, siRNA protein knock-down and in genetically modified mice. In Specific Aim 1, we will generate mice with targeted CFTR deletion in AT1 or AT2 cells and induce cell-specific Ca2+ increases by photolytic uncaging. In Specific Aim 2, we will subject AEC to exogenous H2O2 and alveolar hypoxia. We will determine H2O2 by fluorophore and FRET based imaging. In Specific Aim 3, we will determine the effect of AEC NO on AWL secretion in the context of exogenous NO donors and LPS-induced lung inflammation. Significance: Despite the well-known importance of the AWL in alveolar gas exchange and alveolar immune function, the AWL role in lung inflammation remains poorly understood. Prior to our recent report, no studies addressed AWL formation in the adult lung. For the first time our approach will lead to an understanding of the regulation of AWL formation in the intact lung. These studies will provide new insights into mechanisms underlying the development of acute lung injury. PUBLIC HEALTH RELEVANCE: This project is to determine fundamental mechanisms underlying formation of the alveolar wall liquid by active trans-alveolar chloride transport and its regulation in alveolar hypoxia and lung inflammation. The findings of this research are likely to impact understanding of mechanisms of acute lung injury and the development of relevant therapy.